Proteomic Characterization of Alternative Splicing and Coding Polymorphism Nathan Edwards Center for Bioinformatics and Computational Biology University of Maryland, College Park

Why don’t we see more novel peptides? Tandem mass spectrometry doesn’t discriminate against novel peptides......but protein sequence databases do! Searching traditional protein sequence databases biases the results towards well-understood protein isoforms!

What goes missing? Known coding SNPs Novel coding mutations Alternative splicing isoforms Alternative translation start-sites Microexons Alternative translation frames

Why should we care? Alternative splicing is the norm! Only 20-25K human genes Each gene makes many proteins Proteins have clinical implications Biomarker discovery Evidence for SNPs and alternative splicing stops with transcription Genomic assays, ESTs, mRNA sequence. Little hard evidence for translation start site

Novel Splice Isoform Human Jurkat leukemia cell-line Lipid-raft extraction protocol, targeting T cells von Haller, et al. MCP LIME1 gene: LCK interacting transmembrane adaptor 1 LCK gene: Leukocyte-specific protein tyrosine kinase Proto-oncogene Chromosomal aberration involving LCK in leukemias. Multiple significant peptide identifications

Novel Splice Isoform

Novel Mutation HUPO Plasma Proteome Project Pooled samples from 10 male & 10 female healthy Chinese subjects Plasma/EDTA sample protocol Li, et al. Proteomics (Lab 29) TTR gene Transthyretin (pre-albumin) Defects in TTR are a cause of amyloidosis. Familial amyloidotic polyneuropathy late-onset, dominant inheritance

Novel Mutation Ala2→Pro associated with familial amyloid polyneuropathy

Novel Mutation

Searching Expressed Sequence Tags (ESTs) Pros No introns! Primary splicing evidence for annotation pipelines Evidence for dbSNP Often derived from clinical cancer samples Cons No frame Large (8Gb) “Untrusted” by annotation pipelines Highly redundant Nucleotide error rate ~ 1%

Compressed EST Peptide Sequence Database For all ESTs mapped to a UniGene gene: Six-frame translation Eliminate ORFs < 30 amino-acids Eliminate amino-acid 30-mers observed once Compress to C2 FASTA database Complete, Correct for amino-acid 30-mers Gene-centric peptide sequence database: Size:223 Mb vs 8 Gb, FASTA entries Running time:15 mins vs 22 hours E-values:50-fold reduction Download:

Back to the lab... Current LC/MS/MS workflows identify a few peptides per protein...not sufficient for protein isoforms Need to raise the sequence coverage to (say) 80%...protein separation prior to LC/MS/MS analysis

Future informatics directions... Combine results from multiple searches from multiple engines Fast, automated triage of “significant false-positive” peptide identifications Compressed EST peptide sequence database for other species Mouse, Rat, Zebrafish, Chicken, Cow, A. thaliana, ?? Relational database and web-application infrastructure Interactive browser data-grid, flexible web-services export Java Applet MS/MS viewers, GFF for Genome Browser

Conclusions Peptides identify more than just proteins Untapped source of disease biomarkers Functional vs silencing variants Compressed peptide sequence databases make routine EST searching feasible Statistically significant peptide identification is only the first step

Acknowledgements Catherine Fenselau, Steve Swatkoski UMCP Biochemistry Chau-Wen Tseng, Xue Wu UMCP Computer Science Cheng Lee Calibrant Biosystems PeptideAtlas, HUPO PPP, X!Tandem Funding: NCI